MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Module 5. Techniques for microbiological analyses

Lesson 24
ENUMERATION OF E. COLI/ E. COLI O157:H7

24.1 Introduction

Escherichia coli, originally known as Bacterium coli commune, was identified in 1885 by the German paediatrician, the Theodor Escherich. E. coli is widely distributed in the intestine of humans and warm-blooded animals and is the predominant facultative anaerobe in the bowel and part of the essential intestinal flora that maintains the physiology of the healthy host. E. coli is a member of the family Enterobacteriaceae, which includes many genera, including known pathogens such as Salmonella, Shigella, and Yersinia. Although most strains of E. coli are not regarded as pathogens, they can be opportunistic pathogens that cause infections in immune-compromised hosts. There are also pathogenic strains of E. coli that when ingested, causes gastrointestinal illness in healthy humans.

In 1892, Shardinger proposed the use of E. coli as an indicator of faecal contamination. This was based on the premise that E. coli is abundant in human and animal faeces and not usually found in other niches. Furthermore, since E. coli could be easily detected by its ability to ferment glucose (later changed to lactose), it was easier to isolate than known gastrointestinal pathogens. Hence, the presence of E. coli in food or water became accepted as indicative of recent faecal contamination and the possible presence of frank pathogens. Although the concept of using E. coli as an indirect indicator of health risk was sound, it was complicated in practice, due to the presence of other enteric bacteria like Citrobacter, Enterobacter and Klebsiella that can also ferment lactose and are similar to E. coli in phenotypic characteristics, so that they are not easily distinguished.

Although coliforms were easy to detect, their association with faecal contamination was questionable because some coliforms are found naturally in environmental samples. This led to the introduction of the faecal coliforms as an indicator of contamination. Faecal coliform, first defined based on the works of Eijkman is a subset of total coliforms that grows and ferments lactose at elevated incubation temperature, hence also referred to as thermo tolerant coliforms. Faecal coliform analyses are done at 45.5°C for food testing, except for water, shellfish and shellfish harvest water analyses, which use 44.5°C. The faecal coliform group consists mostly of E. coli but some other enterics such as Klebsiella can also ferment lactose at these temperatures and therefore, be considered as faecal coliforms. The inclusion of Klebsiella spp. in the working definition of faecal coliforms diminished the correlation of this group with faecal contamination. As a result, E. coli has re-emerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.

Currently, all 3 groups are used as indicators but in different applications. Detection of coliforms is used as an indicator of sanitary quality of water or as a general indicator of sanitary condition in the food-processing environment. Faecal coliforms remain the standard indicator of choice for shellfish and shellfish harvest waters; and E. coli is used to indicate recent faecal contamination or unsanitary processing. Almost all the methods used to detect E. coli, total coliforms or faecal coliforms are enumeration methods that are based on lactose fermentation.

24.2 Enumeration and Isolation of E. coli

The Most Probable Number (MPN) method is a statistical, multi-step assay consisting of presumptive, confirmed and completed phases. In the assay, serial dilutions of a sample are inoculated into broth media. Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other two phases of the assay are performed and then uses the combinations of positive results to consult a statistical tables, to estimate the number of organisms present. Typically only the first two phases are performed in coliform and faecal coliform analysis, while all three phases are done for E. coli. The 3-tube MPN test is used for testing most foods. The 5-tube MPN is used for water, shellfish and shellfish harvest water testing and there is also a 10-tube MPN method that is used to test bottled water or samples that are not expected to be highly contaminated.

There is also a solid medium plating method for coliforms that uses violet red bile agar (VRBA) which contains neutral red pH indicator, so that lactose fermentation results in formation of pink colonies. There are also membrane filtration tests for coliform and faecal coliform that measure aldehyde formation due to fermentation of lactose.

24.2.1 Presumptive test for E. coli

Weigh 50 g food into sterile high-speed blender jar. Frozen samples can be softened by storing it for <18 h at 2-5°C, but do not thaw. Add 450 ml of Butterfield's phosphate-buffered water and blend for 2 min. If < 50 g of sample are available, weigh portion that is equivalent to half of the sample and add sufficient volume of sterile diluents to make a 1:10 dilution. The total volume in the blender jar should completely cover the blades.

Prepare decimal dilutions with sterile Butterfield's phosphate diluents. Number of dilutions to be prepared depends on anticipated coliform density. Shake all suspensions 25 times in 30 cm arc or vortex mix for 7 seconds. Do not use pipettes to deliver <10% of their total volume. Transfer 1 ml portions to three LST (Lauryl Sulfate Tryptose) tubes for each dilution for at least three consecutive dilutions. Hold pipette at angle so that its lower edge rests against the tube. Let pipette drain 2-3 seconds. Not more than 15 min should elapse from time the sample is blended until all dilutions are inoculated in appropriate media.

Incubate LST tubes at 35°C. Examine tubes and record reactions at 24 ± 2 hours for gas, i.e. displacement of medium in fermentation vial or effervescence when tubes are gently agitated. Re-incubate gas-negative tubes for an additional 24 hours and examine and record reactions again at 48 ± 2 hours. Perform confirmed test on all presumptive positive (gas) tubes.

24.2.2 Confirmed test

From each gassing LST tube, transfer a loopful of suspension to a tube of BGLB broth, avoiding pellicle if present. Incubate BGLB tubes at 35°C and examine for gas production at 48 ± 2 hours. Calculate most probable number (MPN) (see Appendix 2) of coliforms based on proportion of confirmed gassing LST tubes for three consecutive dilutions.

24.2.3 Confirmed test for faecal coliforms and E. coli by eijkman test

From each gassing LST tube from the presumptive test, transfer a loopful of each suspension to a tube of EC broth (a sterile wooden applicator stick may also be used for these transfers). Incubate EC tubes 24 ± 2 hours at 45.5°C and examine for gas production. If negative, re-incubate and examine again at 48 ± 2 hours. Use results of this test to calculate faecal coliform MPN. The EC broth MPN method may be used for seawater and shellfish since it conforms to recommended procedures

NOTE: Faecal coliform analyses are done at 45.5± 0.2°C for all foods, except for water testing.

24.2.4 Completed test for E. coli

To perform the Completed test for E .coli, gently agitate each gassing EC tube and streak for isolation, a loopful to a L-EMB agar plate and incubate for 18-24 hours at 35°C. Examine plates for suspicious E. coli colonies, i.e., dark cantered and flat, with or without metallic sheen. Transfer up to five suspicious colonies from each L-EMB plate to PCA slants incubate for 18-24 hours at 35°C and use for further testing.

NOTE: Identification of any 1 of the 5 colonies as E. coli is sufficient to regard that EC tube as positive; hence, not all five isolates may need to be tested.

24.2.4.1 Gram stain

All cultures appearing as Gram-negative, short rods should be tested for the IMViC reactions below and also re-inoculated back into LST to confirm gas production.

24.2.4.2 IMViC tests

IMViC reactions are a set of four useful reactions that are commonly employed in the identification of members of family Enterobacteriaceae. The four reactions are: Indole production test, Methyl Red reduction test, Voges Proskauer test and Citrate utilization test.

Indole test

Some bacteria can produce indole from amino acid tryptophan using the enzyme tryptophanase. Production of indole is detected using Ehrlich’s reagent or Kovac’s reagent. Indole reacts with the aldehyde in the reagent to give a red colour. An alcoholic layer concentrates the red colour as a ring at the top.

Procedure

Inoculate tube of tryptone broth and incubate 24 ± 2 hours at 35°C. Test for indole by adding 0.2-0.3 ml of Kovacs' reagent. Appearance of distinct red colour in upper layer is positive test.

Methyl red-reactive compounds

This is to detect the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Some bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system. Methyl Red is a pH indicator, which remains red in colour at a pH of 4.4 or less.

Procedure

Inoculate tube of Glucose peptone water broth and incubate 48 ± 2 hours at 35°C. Transfer 1 ml to 13 x 100 mm tube. Add 5 drops of methyl red solution to each tube. Distinct red colour is positive test. Yellow is negative reaction.

Voges Proskauer (VP) Test

VP test detects butylene glycol producers. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of butylene glycol. In these test two reagents, 40% KOH and alpha-naphthol are added to test broth after incubation and exposed to atmospheric oxygen. If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl. Diacetyl then reacts with guanidine components of peptone, in the presence of α-naphthol to produce red colour. Role of α-naphthol is that of a catalyst and a colour intensifier.

Procedure

Inoculate tube of glucose peptone water broth and incubate 48 ± 2 hours at 35°C. Transfer 1 ml to 13 x 100 mm tube. Add 0.6 ml -naphthol solution and 0.2 ml 40% KOH and shake. Add a few crystals of creatine. Shake and let stand 2 hours. Test is positive if eosin pink colour develops.

Citrate utilization test

This test detects the ability of an organism to utilize citrate as the sole source of carbon and energy. Bacteria are inoculated on a medium containing sodium citrate and a pH indicator bromo-thymol blue. The medium also contains inorganic ammonium salts, which is utilized as sole source of nitrogen. Utilization of citrate involves the enzyme citritase, which breaks down citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and CO₂. Production of Na₂CO₃ as well as NH₃ from utilization of sodium citrate and ammonium salt respectively results in alkaline pH. This results in change of medium’s colour from green to blue.

Procedure

Lightly inoculate tube of Koser's citrate broth; avoid detectable turbidity. Incubate for 96 hours at 35°C. Development of distinct turbidity is positive reaction.

24.2.4.3 Gas from lactose

Inoculate a tube of LST and incubate 48 ± 2 hours at 35°C. Gas production or effervescence after gentle agitation is positive reaction.

24.2.4.4 Interpretation

All cultures that ferment lactose with gas production within 48 hours at 35°C, appear as Gram-negative non spore forming rods Calculate MPN of E. coli based on proportion of EC tubes in three successive dilutions that contain E. coli.

24.2.5 Various media used for the selective isolation of E. coli

24.2.5.1 HiCrome selective ECC base agar

Principle and interpretation

HiCrome ECC Selective Agar is a selective medium recommended for the simultaneous detection of Escherichia coli and total coliforms in water and food samples. The chromogenic mixture contains two chromogenic substrates. The enzyme β-galactosidase produced by coliforms cleaves the chromogen resulting in the salmon to red coloration. The enzyme β-glucuronidase produced by Escherichia coli, cleaves X-glucuronide. Colonies of Escherichia coli are dark blue to violet colored due to cleavage of both the chromogen (Fig. 24.1). The addition of L-tryptophan improves the indole reaction, thereby increasing detection reliability. Cefsulodin, when added inhibits Pseudomonas and Aeromonas species.

Fig. 24.1 Blue colour colonies of E. coli & red colour colonies of E. aerogenes

24.2.5.2 HiCrome M-lauryl sulphate agar

Principle and interpretation

HiCrome M-Lauryl Sulphate Agar is a modification of the Lauryl tryptose broth, formulated by Mallman and Darby. This Chromogenic medium is recommended for the presumptive identification and differentiation of E. coli and other coliforms by a single membrane filtration technique. The incorporation of chromogen X-glucuronide and the dye phenol red favors the differentiation of E. coli and other coliforms on the basis of colour. Peptic digest of animal tissue and yeast extract provide essential growth nutrients to the organisms. Lactose acts as a source of fermentable sugar Sodium Lauryl Sulphate inhibits organisms other than coliforms. The enzyme Beta-glucuronidase produced by E. coli, cleaves X-glucuronide, imparting a green colour to the colonies and along with phenol red indicator aids in detection of lactose fermenter (Fig. 24.2).

Fig. 24.2 Green colour colonies of E. coli are obtained

24.3 Pathogenic E. coli

Shiga toxin producing E. coli (STEC) are food-borne pathogens that may cause serious illness in humans. Among the food related zoonoses, they are the fourth most occurring group in Belgium, but as far as human symptoms are concerned, they are one of the most dreaded organisms. Bovine animals, which are asymptomatic carriers, are the most important reservoir. Infection of humans is mostly caused by the consumption of infected foodstuffs derived from bovine animals, such as milk and milk products.

A wide range of serogroups is capable of provoking these human infections, the most important of them being O26, O103, O111, O145 and O157. The STEC bacteria owes its infective capacity to a combination of virulence properties, the most important of which are: the production of type I and II shiga toxins, which are responsible for kidney failure, the proteins encoded on the LEE locus, responsible for the modification and the intimate adherence to the gastro-intestinal cells, and enterohemolysin, which plays a role in the destruction of blood. The combination of these manifestations results in a complex pathology known as the hemolytic uremic syndrome (HUS). In Belgium, 50 human STEC infections per year are reported on average, 50 percent of which are caused by serogroup O157 and some 20 patients develop HUS. The non-sorbitol-fermenting (NSF) O157 strains have been studied most frequently. In fact, an internationally standardized isolation method for food and feed (ISO 16654) is available for that group and is considered as the gold standard. The method is based upon phenotypic characteristics of the organism, such as increased resistance and enzymatic properties. The NSF O157 strain is isolated in approximately 4 days using conventional culture. First, the sample is selectively enriched by adding antibiotics (novobiocin) for 6 hours at a relatively high temperature (41.5°C). Then, immunomagnetic separation (IMS) is used on the enriched sample.

24.3.1 LST-MUG method for detecting E. coli/E. coli O157:H7

The LST-MUG assay is based on the enzymatic activity of β-glucuronidase (GUD), which cleaves the substrate 4-methylumbelliferyl β-D-glucuronide (MUG), to release 4-methylumbelliferone (MU). When exposed to long wave (365 nm) UV light, MU exhibits a bluish fluorescence that is easily visualized in the medium or around the colonies. Over 95% of E. coli produces GUD, including anaerogenic (non-gas-producing) strains. One exception is enterohemorrhagic E. coli (EHEC) of serotype O157:H7, which is consistently GUD negative. The lack of GUD phenotype in O157:H7 is often used to differentiate this serotype from other E. coli, although GUD positive variants of O157:H7 do exist. The production of GUD by other members of the family Enterobacteriaceae is rare, except for some Shigella (44-58%) and salmonellae (20-29%). However, the inadvertent detection of these pathogens by GUD-based assays is not considered a drawback from a public health perspective. Expression of GUD activity is affected by catabolite repression so on occasion, some E. coli are GUD-negative, even though they carry the uidA gene (gusA) that encodes for the enzyme (19). In most analyses however, about 96% of E. coli isolates tested are GUD-positive without the need for enzyme induction.

MUG can be incorporated into almost any medium for use in detecting E. coli. But some media such as EMB, which contain fluorescent components, are not suitable, as they will mask the fluorescence of MU. When MUG is incorporated into LST medium, coliforms can be enumerated on the basis of gas production from lactose and E. coli are presumptively identified by fluorescence in the medium under longwave UV light, thus it is capable of providing a presumptive identification of E. coli within 24 h.

24.3.2 Presumptive LST-MUG test for E. coli

Prepare food samples and perform the MPN Presumptive test as described in section I.C. above, except use LST-MUG tubes instead of LST. Be sure to inoculate one tube of LST-MUG with a known GUD-positive E. coli isolate as positive control (ATCC 25922). In addition, inoculate another tube with a culture of Enterobacter aerogenes (ATCC 13048) as negative control, to facilitate differentiation of sample tubes that show only growth from those showing both growth and fluorescence. Incubate tubes for 24 to 48 ± 2 h at 35°C. Examine each tube for growth (turbidity, gas) then examine tubes in the dark under longwave UV lamp (365 nm). A bluish fluorescence is a positive presumptive test for E. coli. After 48 h of incubation, 96-100% of E. coli-positive tubes can be identified (28). Perform a confirmed test on all presumptive positive tubes by streaking a loopful of suspension from each fluorescing tube to L-EMB agar and incubate 24 ± 2 h at 35°C. Follow protocols outlined in 24.2.4, above, for Completed test for E. coli. Calculate MPN of E. coli based on combination of confirmed fluorescing tubes in 3 successive dilutions.